PROJECT SUMMARY Ovarian cancer is the 5th leading cause of cancer death in women. The overall 5-year survival rate for epithelial ovarian cancer (EOC), the most common type, remains approximately 30% over the last several decades. High- grade serous ovarian cancer (HGSOC) is the most common and lethal form of EOC. Thus, the novel strategies for treating HGSOC are urgently needed. Cancer cells often carry genetic defects that provide opportunities for specific targeting. Expression of the B55 ?, a regulatory subunit of the heterotrimeric serine/threonine phosphatase PP2A, is reduced in 46% of ovarian cancers, as a result of loss of heterozygosity. Our recent novel genome-wide synthetic lethality screen discovered that B55 ? reduction led to the enhanced sensitivity of cells to an inhibitor targeting replication stress response kinase CHK1. Currently, inhibitors of CHK1 and its upstream effector ATR are being tested in clinical trials without precise guidance of biomarkers to identify responsive populations, which significantly reduce the efficacy of these agents. Thus, the goal of this application is to establish the molecular basis for targeting B55 ? defective HGSOC by ATR/CHK1 inhibitors and for B55 ? being a biomarker to guide the use of those agents. PP2A has diverse functions, including the negative regulation of numerous oncogenic signaling. Oncogene activation can cause replication stress that could be lethal to the cells. ATR/CHK1 signaling suppresses replication stress to less toxic levels. Thus, cancer cells with increased replication stress, such as cancer cells with c-Myc activation/expression, heavily rely on ATR/CHK1 for survival. Given the role of PP2A in negatively regulating multiple oncogenic pathways, B55 ? deficiency may lead to oncogene activation and replication stress, rendering these cells sensitive to ATR/CHK1 inhibition. We hypothesize that ATR and CHK1 inhibitors specifically target B55 ?-defective HGSOC cells by enhancing replication stress. Thus, reduced B55 ? expression could be a biomarker to guide the use of ATR and CHK1 inhibitors. Three Specific Aims are proposed. In Aim 1, we will determine the role of c-Myc in B55 ? deficiency- induced replication stress. In Aim 2, we will determine the causes of the B55 ? deficiency-induced replication stress. In Aim 3, we will assess the anti-tumor activities of ATR and CHK1 inhibition in targeting B55 ? defective HGSOC cells. If successful, our study will have a significant impact on improving the survival of ovarian cancer patients by identifying new treatment approaches and by providing a new stratification biomarker for identifying ATR/CHK1 inhibitor sensitive populations.